It ordered the diplomats to leave within a week.
Britain's foreign secretary accused Russian President Vladimir Putin of personally ordering the poisoning of the Skripals.
"We think it overwhelmingly likely that it was his decision to direct the use of a nerve agent on the streets of the United Kingdom, on the streets of Europe, for the first time since the World War 2", Johnson said in London. Putin's spokesman denounced the claim. U.K. Foreign Minister Boris Johnson said it was the first instance of nerve agents being used in Europe since World War II.
Russia's response does not change the fact that Moscow was in "flagrant breach of worldwide law" and the Chemical Weapons Convention, she said.
Russian Foreign Ministry spokeswoman Maria Zakharova denied that Russia or the Soviet Union had ever developed Novichok, the class of nerve agent that Britain says was used to poison the Skripals.
But she said Britain had "no disagreement with the Russian people". A confirmation was given by the police that Mr Skripal was a British citizen.
UK-Russia relations have been fractious ever since the assassination of another former Russian spy, Alexander Litvinenko, in 2006.
The diplomatic standoff started with the poisoning of former Russian double agent Sergei Skripal and his daughter, Yulia, in Salisbury, England on March 4. On Saturday, Russia's envoy to the Organisation for the Prohibition of Chemical Weapons (OPCW), Alexander Shigulin alleged to the news agency Interfax the "most likely" source of the nerve agent was Britain or the United States.
Russian Federation has declared members of the British diplomatic mission persona non grata.
"When accusations are being made against a country, our country, in this case, very serious allegations, formulated through the use of "evidently, ' 'highly likely" and so on", Peskov said.More news: Egyptians living overseas open presidential poll
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Separately on Friday, police in London launched a murder investigation into the death this week of a Russian associate of late tycoon Boris Berezovsky, but said nothing at this stage suggested a link to the Skripal case.
Russian Federation has also said it is opening a probe into the murder of Glushkov, who was found dead at his London home.
A post-mortem examination found the 68-year-old died from "compression to the neck".
Putin has barely weighed in on the row, telling a BBC reporter this week: "Sort things out from your side and then we will discuss this with you".
The Russian Foreign Ministry summoned the British ambassador, Laurie Bristow, to a meeting on Saturday morning in central Moscow at headquarters during which he was informed of the measures.
Skripal had taken his daughter, who was on a visit from Moscow, out for lunch in Salisbury before they both collapsed on a bench.
Many Russians remain sceptical that the state was responsible and some analysts have not ruled out the involvement of ordinary criminals or rogue agents.
The UK ordered Russian diplomats to leave the country over the incident on 4 March which the UK government has blamed on Russia - but which Russia denies.
In London, opposition Labour Party leader Jeremy Corbyn struck a starkly different tone to that of the British government by warning against rushing into a new Cold War before full evidence of Moscow's culpability was proven.
"Due to the unregulated status of the British Council in Russian Federation, its activity is halted", the foreign ministry said.